Effects of TDP2/VPg Unlinkase Activity on Picornavirus Infections Downstream of Virus Translation
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture and Virus Stocks
2.2. Generation of 35S-Methionine-Labeled VPg-Linked RNA Substrate and VPg Unlinkase Assay
2.3. Generation of Tagged TDP2-Expressing hRPE Cell Lines
2.4. Virus Infections and Single Cycle Growth Analysis
2.5. Preparation of Lysates from Uninfected and Infected Cells and Western Blot Analysis
2.6. Generation of Renilla Luciferase Reporter Virus and Luciferase Assays
2.7. Polysome Profile Analysis and Quantitation of Viral RNA Production
2.8. Encapsidation Inhibition Assays
2.9. Immunofluorescence Assays
3. Results
3.1. TDP2 Protein Expression and VPg Unlinkase Activity Are Absent in TDP2 Knock-Out hRPE-1 Cells
3.2. Virus Growth Kinetics Are Delayed in the Absence of TDP2 for Poliovirus and CVB3 in hRPE-1 Cells, and the Effect is MOI-Dependent
3.3. Poliovirus Titers Are Comparable in WT and KO RPE cells after Multiple Replication Cycles
3.4. eIF4G is Cleaved in both WT and KO TDP2 RPE Cells during Poliovirus Infection
3.5. An Alternative Source of VPg Unlinkase Activity Is Not Activated at Late Times of Infection in TDP2 KO hRPE-1 Cells
3.6. TDP2 5′ Phosphodiesterase Activity Is Required for Efficient Poliovirus Replication
3.7. Poliovirus Protein Production Is Delayed in the Absence of TDP2 in hRPE-1 Cells
3.8. Polysomes Can Assemble Efficiently on VPg-Linked RNA after the Initial Round of Translation and Replication in Cultured Cells
3.9. Viral RNA Synthesis Is Delayed in the Absence of TDP2 in hRPE Cells
3.10. Premature Encapsidation of Viral RNA Is not Responsible for Observed Growth Phenotype in the Absence of TDP2
3.11. Replicative Form (RF) and/or Replicative Intermediate (RI) Formation Is Delayed in TDP2 KO hRPE-1 Cells
4. Discussion
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Holmes, A.C.; Zagnoli-Vieira, G.; Caldecott, K.W.; Semler, B.L. Effects of TDP2/VPg Unlinkase Activity on Picornavirus Infections Downstream of Virus Translation. Viruses 2020, 12, 166. https://doi.org/10.3390/v12020166
Holmes AC, Zagnoli-Vieira G, Caldecott KW, Semler BL. Effects of TDP2/VPg Unlinkase Activity on Picornavirus Infections Downstream of Virus Translation. Viruses. 2020; 12(2):166. https://doi.org/10.3390/v12020166
Chicago/Turabian StyleHolmes, Autumn C., Guido Zagnoli-Vieira, Keith W. Caldecott, and Bert L. Semler. 2020. "Effects of TDP2/VPg Unlinkase Activity on Picornavirus Infections Downstream of Virus Translation" Viruses 12, no. 2: 166. https://doi.org/10.3390/v12020166
APA StyleHolmes, A. C., Zagnoli-Vieira, G., Caldecott, K. W., & Semler, B. L. (2020). Effects of TDP2/VPg Unlinkase Activity on Picornavirus Infections Downstream of Virus Translation. Viruses, 12(2), 166. https://doi.org/10.3390/v12020166